Generating cardiovascular cells from pluripotent stem cells holds great promise for cardiovascular research and therapy. The past 10 years have seen rapid methodological advances for creating de novo human cardiomyocytes. In particular, the ability of human pluripotent stem cells (hPSCs) to differentiate to cells in cardiac lineages has attracted significant interest, with a strong focus on applications in modeling cardiovascular development, drug discovery, safety pharmacology and cell therapy in myocardial infarction and heart failure.
Several efficient differentiation strategies involving stage-specific activation and inhibition of different signaling pathways, with the goal of mimicking in vivo cardiac development, have been devised to generate cardiomyocytes from hPSCs (see, e.g., Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524-8 (2008); Kattman, S. J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228-40 (2011); Laflamme, M. A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015-24 (2007); Burridge, P. W. et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One 6, e18293 (2011); and Mummery, C. et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733-40 (2003)). However, most of these approaches are not chemically-defined, contain xenogenic components, or require expensive recombinant proteins, limiting their application for large-scale cardiomyocyte production for therapeutic applications.
Previously, we disclosed the Gsk3 inhibitor/Wnt inhibitor (GiWi) method for robust hPSC differentiation to cardiomyocytes under serum-free and growth factor-free conditions (see U.S. Patent Publication No. 2013/0189785, which is incorporated by reference herein). In one embodiment, this GiWi method applies two small molecules, a Gsk3 inhibitor and a Porcupine inhibitor, at precise developmental stages to sequentially promote mesoderm formation and then cardiomyocyte specification (Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162-75 (2013); Lian, X. J. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. U.S.A. 109, E1848-E1857 (2012)). Although the RPMI/B27-ins (B27 without insulin) medium used in the GiWi protocol lacks animal sera and growth factors, the inclusion of bovine serum albumin (BSA) increases the cost and adds xenogenic components, limiting the protocol's potential for large-scale cardiomyocyte production. Furthermore, the use of BSA in the GiWi protocol may decrease differentiation consistency, because of the substantial batch-to-batch variability of the albumin used in the protocol.
Despite these known disadvantages of using albumin in protocols for differentiating human pluripotent stem cells to cardiomyocytes, albumin is an essential ingredient in all previously disclosed protocols. In some protocols, the BSA is replaced with a recombinant albumin, such as recombinant human albumin (see, e.g., U.S. Patent Publication No. 2014/0134733). Although the use of recombinant albumin may address some of the issues associated by the use of albumin in differentiation protocols (e.g., the need for batch-to-batch consistency resulting from impurities in animal-derived albumin), it is substantially more costly than BSA, and its use would be prohibitively expensive for a commercially viable protocol optimized for large-scale cardiomyocyte production.
Accordingly, there is a need in the art for a cardiac differentiation protocol that uses defined culture conditions in the absence of albumin or other substances previously thought to be an essential part of such protocols to produce cardiomyocyte progenitors and cardiomyocytes from hPSCs in a more efficient, cost-effective manner.